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Regeneration of tooth germ using dental epithelial stem cells and dental papilla mesenchymal stem cells in rodent incisors

利用牙上皮干细胞和牙乳头间充质干细胞在啮齿动物门牙中再生牙胚

基本信息

批准号：
16390527
负责人：
HARADA Hidemitsu
金额：
$ 8.7万
依托单位：
Iwate Medical University (2006)Osaka University (2004-2005)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

项目摘要

Mouse incisors are regenerative tissues, which grow continuously throughout life. We found that in the teeth, Fgf-9 plays a role of maintenance of mesenchymal cells expressing Fgf-10 and that the stem cell niche of incisors are formed by the epithelial-mesenchymal interaction via the signaling of Fgf-9 and Fgf-10. Immunostaining showed that Fgf-9 was expressed in the basal epithelium, stellate reticulum and inner enamel epithelium in the apical bud, and the expression area underlay the mesenchyme expressing Fgf-10. Recombinant Fgf-9 protein stimulated the increase of number of mesenchymal cells in a concentration-dependent manner. Annexin V staining and whole mount in situ hybridyzation using organ culture showed that recombinant Fgf-9 protein inhibited the apoptosis of mesenchymal cells of apical end and maintained the expression of Fgf-10. However, we could not success to produce bioengineered tooth germ and to culture these stem cells in the presence of only these factors.Furthermore, we investigated the additionally factors in the culture medium and it was found that we needs FGF2 and EGF to culture the cells of the epithelial and mesenchymal stem cell compartment.Finally, we succeeded to produce the regenerative bioengineered teeth using the dental epithelial stem cells and the mesenchymal stem cells of mouse incisors by the combination of collagen sponge and Fgf2/Egf/Fgf9/Fgf10-releasing gelatin beads. Now, we proceed to study the technical methods controlling the morphology and size of bioengineered tooth by these factors.

小鼠门牙是再生组织，在整个生命中不断生长。我们发现，在牙齿中，Fgf-9起着维持表达Fgf-10的间充质细胞的作用，并且切牙的干细胞龛是通过Fgf-9和Fgf-10的信号传导通过上皮-间充质相互作用形成的。免疫组化显示，Fgf-9表达于顶芽的基底上皮、星网状上皮和内釉上皮，表达区位于表达Fgf-10的间充质周围。重组Fgf-9蛋白以浓度依赖性方式刺激间充质细胞数量的增加。Annexin V染色和器官培养整体原位杂交结果显示，重组Fgf-9蛋白可抑制根尖间充质细胞的凋亡，并维持Fgf-10的表达。然而，我们不能成功地生产生物工程牙胚和培养这些干细胞在只有这些因素的存在下。此外，我们调查了培养基中的其他因素，发现我们需要FGF 2和EGF来培养上皮和间充质干细胞室的细胞。最后，我们利用小鼠切牙的牙上皮干细胞和间充质干细胞，通过胶原海绵和Fgf 2/Egf/Fgf 9/Fgf 10的组合，成功地制备了再生生物工程牙齿。释放明胶珠。本课题研究通过这些因素控制生物工程牙的形态和大小的技术方法。